INTRODUCTION:

Transdermal therapeutic systems are designed for controlled drug delivery through the skin into systemic circulation maintaining consistent efficacy and reducing dose of the drug and its related side effects^1,2. It also improves patient compliance, safety and efficacy of the drug^3,4. Propranolol hydrochloride is used in the treatment of angina pectoris, cardiac arrhythmia and hypertension. It is the drug of choice for sustained release formulation since it has a low terminal elimination half life of about 3 to 5 h, which requires frequent dosing necessary to maintain the therapeutic blood level for a long term treatment. The drug shows considerable first pass metabolism and there by has poor bioavailability (15 - 23%) when administered orally^{5, 6} and the low molecular weight (295.81) of the drug again indicates it’s suitability for administration by the transdermal route. Hence the objective of the present study deals with the development and preparation of Propranolol hydrochloride transdermal films using 12 different combinations of the three polymers namely ethyl cellulose (EC), polyvinyl pyrrolidone (PVP) and HPMC by solvent

evaporation technique by keeping the concentration of drug at 20 % w/w constant. All prepared formulations were characterized and in vitro data were correlated with the in vivo datas obtained by transdermally administering to rabbits.

MATERIALS AND METHOD

Ethyl cellulose (EC-20cps), polyvinyl pyrrolidone (PVP-K30), hydroxyl propyl methyl cellulose (HPMC-K4M), polyvinyl alcohol (PVA) and dibutyl phthalate were procured from S.D. Fine Chem. Ltd. Mumbai. Propranolol hydrochloride was received as generous gift sample from Sun Pharmaceuticals Ltd., Baroda, Gujarat. All other chemicals and solvents used were of analytical grade.

Preparation of the Transdermal Patches

Matrix type transdermal patches containing propranolol hydrochloride were prepared using three polymers in two combinations and in different proportions like EC with PVP K30 and EC with HPMC K4M by solvent evaporation technique using glass mould. The backing membrane was cast by pouring 4 % w/v PVA solution in distilled water followed by drying at 60°C for 6 hours in an oven⁹. The polymers of each combination were dissolved in chloroform. Dibutyl phthalate (30 % w/w) of polymer composition was added as plasticizer. Propranolol hydrochloride at a concentration of 20 % w/w of polymer was added and stirred with a mechanical stirrer to get a homogeneous dispersion. The dispersion (2 ml) was cast on the prepared PVA backing membrane in each mould. The rate of evaporation was controlled by inverting a funnel over the mould and dried at 40 °C for 6 hours in hot air oven and stored in desiccators for further use.

Evaluations of transdermal patches

a) Moisture content study

The prepared films were weighed and kept in desiccator containing activated silica at room temperature for 24 hours. The individual films were weighed on every alternate day until a constant weight was achieved. The percentage of moisture content was calculated by determining the difference between initial and final weight with respect to final weight¹⁰.

b) Determination of Folding endurance¹¹

Folding endurance of the film was determined manually by folding a small strip of the film at the same place till it breaks. The maximum number of folding operation done at the same place of the film without breaking, gives the value of folding endurance, where the cracking point of the films were considered as the end point.

c) Water vapor transmission (WVT) rate study¹³

The method is followed as like as per the reference Ragavendra et al. This study involves glass vials of equal diameter (1.4 mm) as transmission cells. The transdermal patch of known thickness was fixed over the edge of the glass vial containing 3 gm of fused calcium chloride as a desiccant by using an adhesive. The initial weight of cells were measured and kept in a desiccator containing saturated solution of potassium chloride (200 ml), which provide the higher RH. The cells were verified regarding weight periodically over a period of 72 hours. Calculations are made by this formula,

WVT rate = W*L / S …………………. (1)

Where, W is water vapour transmitted in gm., L is thickness of the transdermal patch (cm.) and S is exposed surface area (cm²).

d) Elongation and tensile strength measurement

The Tensile strength measurement was made using an instrument assembled in the laboratory and following the method used by Sadhana et al ¹⁴. The films were fixed individually to the assembly, the required weights to break the films were noted. Percentage of elongation of the films was measured by attaching a pointer mounted on the assembly. Tensile strength was calculated by using the following formula,

Tensile strength = (break force/a × b) × (1+L/I)…. (2)

Where, a, b, L and I are the width, thickness, length and elongation of the films respectively.

Table-1: Formulation Design of the Propranolol Transdermal Patches.

Formulation code	Drug + Ratio of polymers (EC: PVP)	Formulation code	Drug +Ratio of polymers (EC: HPMC)
F1	1:2	F7	1:2
F2	1:4	F8	1:4
F3	1:6	F9	1:6
F4	2:1	F10	2:1
F5	4:1	F11	4:1
F6	6:1	F12	6:1

*Concentration of drug was maintained at 20 % w/w for all the formulations.

F1 = EC-PVP (1:2) F7 = EC-HPMC (1:2)

F2 = EC-PVP (1:4) F8 = EC-HPMC (1:4)

F3 = EC-PVP (1:6) F9 = EC-HPMC (1:6)

F4 = EC-PVP (2:1) F10 = EC-HPMC (2:1)

F5 = EC-PVP (4:1) F11 = EC-HPMC (4:1)

F6 = EC-PVP (6:1) F12 = EC-HPMC (6:1)

e) In vitro skin permeation study ^{15, 16}

In vitro skin permeation study was performed by using albino rat skin. The weighed Young albino rat (200 gm - 250 gm) were taken and sacrificed. The abdominal skin was carefully separated from the body after abdominal hairs were removed. The skin was treated with 0.32 M ammonia solution for 35 m. The skin, so obtained, was examined microscopically for any possible damage. The full thickness skin thus obtained was kept in normal saline solution and stored at (4 ± 1)°C. The drug permeation from the transdermal patches through the skin was determined using modified Keshary-Chien diffusion cell filled with 100 ml of phosphate buffer of pH 7.4 maintaining temp at (37 ± 1)°C stirring continuously at a constant speed of 100 rpm. At a time interval of 1 hr samples of 1 ml were withdrawn replaced by fresh buffer over a period of 48 hours. The samples were then analyzed spectrophotometrically at λ _max 290 nm.

f) In vitro drug release kinetics ¹⁷

The patterns of drug release and exact mechanism of drug release from different transdermal patches was studied by applying different kinetic model like higuchi square root and korsemeyer peppas model. The criterion for selecting the most appropriate model was chosen on the basis of goodness of fit test.

g) In vivo experiment ^{18, 19}

Animal Experiment: This study was carried out in healthy male New Zealand albino rabbits (1.5 Kg). Four rabbits were fasted overnight and the dorsal surface was cleaned and hair was removed. During study period animals were kept in aseptic condition.

Analytical Methodology: This study was carried out in a parallel design. Transdermal patches were placed on to the clean dorsal surface occluded with an adhesive tape. Blood samples of 1 ml were withdrawn from the marginal ear vein at 1, 2, 4, 8, 12, 16, 24, 36 and 48 hours using a syringe

Table-2: Study of various physical parameters of the drug loaded transdermal patches composed of EC-PVP and EC – HPMC.

Formulation code.	Ratio of polymers (EC:PVP)	Moisture content (%) (X±S.D.)	Folding endurance (X±S.D.)	Water vapor transmission rate (gm/cm/h) (X±S.D.)	Tensile strength (gm/cm²) (X±S.D.)	*In vitro* drug release (%) in 48 hours (X±S.D.)
F1	1:2	1.04±0.012	261 ± 1.26	1.01754 × 10 ^-4	251.36±0.032	83.82±0.12
F2	1:4	1.09±0.01	208 ± 1.41	1.12437 × 10 ^-4	243.67±0.042	88.81±0.13
F3	1:6	1.13±0.04	183 ± 1.31	1.56959 × 10^-4	221.54±0.012	91.63±0.17
F4	2:1	0.68±0.03	202 ± 1.11	1.30719 × 10 ^-4	253.52±0.022	65.73±0.12
F5	4:1	0.46±0.07	211 ± 1.12	1.27758 × 10 ^-4	257.60±0.032	62.65±0.19
F6	6:1	0.39±0.04	220 ± 1.04	1.21636 × 10 ^-4	261.48±0.062	56.33±0.11
	EC:HPMC
F7	1:2	1.14±0.03	149 ± 1.73	1.33853 × 10 ^-4	234.45±0.032	93.33±0.17
F8	1:4	1.18±0.01	143 ± 2.14	1.47524 × 10 ^-4	221.67±0.012	91.86±0.19
F9	1:6	1.21±0.03	142 ± 1.93	1.85419 × 10 ^-4	204.12±0.072	95.19±0.21
F10	2:1	0.88±0.04	151 ± 1.43	1.10648 × 10 ^-4	238.04±0.112	78.53±0.19
F11	4:1	0.66±0.09	153 ± 1.76	1.10472 × 10 ^-4	237.16±0.312	70.61±0.20
F12	6:1	0.59±0.06	196 ± 1.03	1.09766 × 10 ^-4	256.21±0.042	65.58±0.23

All the values are expressed as mean ± Standard deviation (n=3).

Standard error mean < 1.21. All data are found as statistically significant at 5 % level of significance.

ANOVA
Source of Variation	SS	df	MS	F	P-value	F crit
Between Groups	562178	4	140544.5	364.7035	7.03601E-39	2.539689
Within Groups	21195.16	55	385.3665
Total	583373.2	59

Fig 1. The moisture content of all Transdermal patches formulations.

F1 = EC-PVP (1:2) F7 = EC-HPMC (1:2)

F2 = EC-PVP (1:4) F8 = EC-HPMC (1:4)

F3 = EC-PVP (1:6) F9 = EC-HPMC (1:6)

F4 = EC-PVP (2:1) F10 = EC-HPMC (2:1)

F5 = EC-PVP (4:1) F11 = EC-HPMC (4:1)

F6 = EC-PVP (6:1) F12 = EC-HPMC (6:1)

containing 3.8 % sodium citrate to prevent clotting. The plasma was separated immediately by centrifugation at 2000 rpm for 10 min and stored in the refrigerator until analysis. The drug concentration in the samples was measured by HPLC (shimadzu SPD 20). The experiment was carried out in triplicate.

Fig 2. In vitro drug permeation study of transdermal patch formulations.

F1 = EC-PVP (1:2) F2 = EC-PVP (1:4)

F3 = EC-PVP (1:6) F4 = EC-PVP (2:1)

F5 = EC-PVP (4:1) F6 = EC-PVP (6:1)

h) Scanning Electron Microscopy (SEM)

The SEM analysis was carried out using a scanning electron microscope (LEO, 435 VP, U.K.). Prior to examination, samples were mounted on an aluminium stub using a double sided adhesive tape and making it electrically conductive by coating with a thin layer of gold (approximately 20 nm) in vacuum. The scanning electron microscope was operated at an acceleration voltage of 15 kV²⁰.

i) Statistical analysis

All the data were statistically verified with standard deviation, standard error mean and one way ANOVA at 5 % level of significance ²¹.

Fig 3. In vitro drug permeation study of transdermal patch formulations.

F7 = EC-HPMC (1:2) F8 = EC-HPMC (1:4)

F9 = EC-HPMC (1:6) F10 = EC-HPMC (2:1)

F11 = EC-HPMC (4:1) F12 = EC-HPMC (6:1)

Fig 4. Higuchi square root plot of Transdermal patch formulation F6, EC-PVP (6:1).

RESULTS AND DISCUSSION:

Twelve set (F1-F12) of transdermal patches were prepared and evaluated accordingly. The formulation design and ratio was presented in Table-1. The lipophilic and hydrophilic polymer concentrations are increased and decreased for both PVP and HPMC polymer. Lipophilic polymer used was Ethylcellulose. The concentration of drug is kept constant for every formulation. The moisture content of all the formulations was shown in Table - 2 and fig-1. The moisture content is increased as hydrophilic polymer concentration increased and similarly decreased as hydrophilic concentration increased. Among all the formulations the lowest moisture content was found in F6, which facilitate the better preservation property, and limits the bulkiness of the patches. Folding endurance of all the formulations were found to be varied in between 142 to 261 as narrated in Table-2. It was found that films with higher proportion of PVP and HPMC showed drastic reduction in film endurance in comparison to EC variation. It is evident from the result that higher the EC proportion more was the film endurance. Formulations F6 with drug and polymer ratio 6:1 (EC: PVP) showed an optimum endurance of 220 which prove its efficacy. Weight variations in the formulated patches were found to be very less. Thus formulations F4, F5, F6, F10, F11 and F12 have shown lesser affinity towards water in comparison to other formulations shown in Table-2. Patches containing EC with PVP and EC with HPMC showed a water vapour transmission profile within the range of 1.01754 × 10^-4gm/cm/h to 1.56959 × 10^-4gm/cm/h and 1.09766 × 10 ^-4gm/cm/h to 1.85419 × 10^-4gm/cm/h respectively. It was found that amongst all the formulations F9 has shown the least water vapour transmission profile may be due to having lesser concentrations of PVP with highest concentration of hydrophobic polymer EC. With the increase in the proportions of PVP and HPMC in the films, the tensile strength of the films was found to be significantly decreased as shown in Table-2. The variation in percentage elongation was found to be insignificant over the different proportions of the polymers used. Formulation F6 showed a less percentage elongation and high tensile strength in comparison to other formulation. All the optimized formulation released almost 60 % of drug within 48 hours represented in Table-2. A comparative study of % cumulative drug release Vs. time graph for all the formulations were shown in fig-2 and fig -3. F6 was shown the best controlled release among all the formulation 56.33 % in 48 hours as shown in Table-2.

Fig 5. Korsemeyer and Peppas plot of Transdermal patch formulation F6, EC-PVP (6:1).

Fig 6. In Vivo drug permeation study of Transdermal patch formulation F6, EC-PVP (6:1).

Fig 7. The photomicrographs of scanning electron microscopy (SEM) of transdermal patch before skin permeation study at resolution 15 kV × 1,500.

Fig 8. The photomicrographs of scanning electron microscopy (SEM) of transdermal patch after skin permeation study at resolution 15 kV × 2,000.

The data obtained from the in vitro permeation study of final selected formulation F6 was fitted to the kinetic models (Higuchi and Korsmeyer-peppas model) to determine the pattern of drug release from the drug-polymer matrix as given in fig 7 and 8. Increasing in the concentration of hydrophilic polymer in the transdermal patches would result in higher amount of drug release within lesser time period discussed in Table-2. The F6 formulation among all other formulations was found to be best fitted in Higuchi square root model that confirms its release by diffusion method. Applying one way ANOVA all the data were found to be significant (F=364.7035) at 5 % level of significance. The formulation F6 was selected for in vivo drug permeation study on the basis of its in vitro evaluation results. White New Zealand Male healthy rabbits were used for this study. Data obtained after analyzing the samples by HPLC was plotted against plasma concentration (ng/ml) Vs time. From the graph it was noted that at 24 hours the concentration of the drug was 14.2791 ng/ml, which extends up to 48 hours giving a concentration of 14.7992 ng/ml of the formulation as shown in fig - 6. Drug concentrations raised during the initial hours of the formulation which is the predominant requirement for the success of transdermal formulations. The photomicrographs of scanning electron microscopy (SEM) of before skin permeation study and after skin permeation study was presented in fig-7 and 8 respectively.

CONCLUSION:

In conclusion, extent of propranolol hydrochloride release from its transdermal patches showed that the films containing higher proportion of PVP F6 (6:1 ratio) suitable for once a day drug delivery and the films containing higher proportion of EC showed suitability for a prolonged regimen of sustained drug delivery through transdermal route for a period of more than 48 h. The out comes of the current experiment advocates a rational guideline for formulating a sustained release transdermal therapeutic system of propranolol hydrochloride for effective therapy and prophylaxis of angina pectoris, cardiac arrhythmia and hypertension.

ACKNOWLEDGEMENT

Authors wish to thank Sun Pharmaceuticals Ltd., Baroda, Gujarat, for providing gift sample of Propranolol. Authors wish to thank S.D. Fine chemicals, Mumbai, for providing polymers and chemicals.

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Received on 24.10.2008 Modified on 17.12.2008

Research J. Pharm. and Tech. 2(1): Jan.-Mar. 2009; Page 155-160